Abstract

Breast cancer is the second most common cancer among women in the US. Genetics, environment, as well as dietary factors are thought to have significant impacts on breast cancer risk. Epidemiological evidence supports a role for vitamin D in protection against breast cancer. 1,25 dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin D, is proposed to regulate cellular processes that are involved in breast cancer progression. Metabolic reprogramming of glucose, termed the Warburg effect, as well as increased glutamine uptake and metabolism for energy use is a characteristic of many cancer cells. The current studies were designed to investigate the effect of 1,25(OH)2D on glutamine metabolism in mammary cells during an early stage of cancer progression. We employed untransformed (MCF10A) and ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Intracellular glutamine and glutamate levels, determined by nuclear magnetic resonance, were both reduced with 1,25(OH)2D by 23% in MCF10A-ras cells. However, 1,25(OH)2D decreased intracellular glutamine level in MCF10A cells by only 9%, with no effect on intracellular glutamate level in MCF10A cells. Glutamine and glutamate flux into the TCA cycle were determined using [U-13C5] L-glutamine and gas chromatography-mass spectrometry. Treatment with 1,25(OH)2D decreased glutamine flux into the TCA cycle by 18% and 31% in MCF10A and MCF10A-ras cells, respectively, and decreased glutamate flux into the TCA cycle by 13% and 17% in MCF10A and MCF10A-ras cells, respectively. The mRNA and protein expression of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D in MCF10A-ras cells. Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D, and the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA) inhibited the effect of 1,25(OH)2D on glutamine uptake in both MCF10A and MCF10A-ras cells. The mRNA level of the glutamine transporter, SLC1A5, was also decreased by 1,25(OH)2D in MCF10A which overexpress ErbB2 (HER-2/neu) and in malignant MCF10CA1a cells. These results suggest 1,25(OH)2D may alter glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. The 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may play a role in vitamin D prevention of breast cancer.